Intra- and interindividual epigenetic variation in human germ cells.

Epigenetics represents a secondary inheritance system that has been poorly investigated in human biology. The objective of this study was to perform a comprehensive analysis of DNA methylation variation between and within the germlines of normal males. First, methylated cytosines were mapped using bisulphite modification-based sequencing in the promoter regions of the following disease genes: presenilins (PSEN1 and PSEN2), breast cancer (BRCA1 and BRCA2), myotonic dystrophy (DM1), and Huntington disease (HD). Major epigenetic variation was detected within samples, since the majority of sperm cells of the same individual exhibited unique DNA methylation profiles. In the interindividual analysis, 41 of 61 pairwise comparisons revealed distinct DNA methylation profiles (P=.036 to 6.8 x 10(-14)). Second, a microarray-based epigenetic profiling of the same sperm samples was performed using a 12,198-feature CpG island microarray. The microarray analysis has identified numerous DNA methylation-variable positions in the germ cell genome. The largest degree of variation was detected within the promoter CpG islands and pericentromeric satellites among the single-copy DNA fragments and repetitive elements, respectively. A number of genes, such as EED, CTNNA2, CALM1, CDH13, and STMN2, exhibited age-related DNA methylation changes. Finally, allele-specific methylation patterns in CDH13 were detected. This study provides evidence for significant epigenetic variability in human germ cells, which warrants further research to determine whether such epigenetic patterns can be efficiently transmitted across generations and what impact inherited epigenetic individuality may have on phenotypic outcomes in health and disease.

Structure-based ligand binding sites of protein p14.5, a member of protein family YER057c/YIL051c/YjgF.

Seventeen mutants with one, two or three amino acids substitutions of human protein p14.5, homologue to well-known tumor antigen from goat liver UK114 and a member of proteins YER057c/YIL051c/YjgF family, have been used for structure-functional relation studies and ligand binding analysis using cross-linking by triacryloyl-hexahydro-s-triazine (TAT), size exclusion chromatography, free fatty acid and 8-anilino-1-naphthalenesulfonic acid (ANS) binding assays. Amino acids having the most significant impact on the ligand binding activity have been determined: R107, N93, Y21 and F89. Arginine 107 has been identified as the most accessible amino acid in the cleft. Trimeric structure of protein p14.5 has been confirmed as being essential for stoichiometric small ligand binding activity and oligomeric structure of p14. Ligand binding activity may be related with the biological functions of these proteins, which still are not understood well.

DNA methylation differences in human p14.5 gene promoter region in normal and proliferating cells.

Methylation status of cytosines and its changes during cell proliferation was analyzed in the 5'-flanking region of the human p14.5 gene, which encodes a member of the YER057c/YIL051c/YjgF protein family. We describe evidence of dramatic DNA methylation differences revealed in the study, and present detailed mapping of methylated cytosines (metC) at the 5'-flanking region of the p14.5 gene in several human normal tissues and tumor cells lines. DNA methylation profiles demonstrated aberrant distribution of metC positions with the different degree of methylation along all analyzed 5'-flanking regions of the p14.5 gene in cancer cells. We investigated DNA methylation changes in p14.5 5'-flanking region during cell differentiation by using DNA samples of freshly isolated monocytes and macrophages. According to our data, cellular differentiation processes from monocytes to macrophages are related to the elevated degree of DNA methylation of the p14.5 gene at the putative binding motifs for several transcription factors. The present findings indicate that some cytosines in the promoter region may have some significance in the degree of expression of the p14.5 gene during cell proliferation and cancerogenesis.

Identification of differentially expressed genes in yeast Saccharomyces cerevisiae cells with inactivated Mmf1p and Hmf1p, members of proteins family YERO57c/YJGF.

We used differential display analysis of mRNA to investigate the differences between gene expression in wild-type (wt) yeast Saccharomyces cerevisiae cells and mutated ones with disrupted activity of genes MMF1 and HMF1, members of the YERO57c/YJGF family. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to determine the differences in the degree of expression of 14 specific transcripts in normal and mutated yeast cells. Obtained data demonstrate that disruption of genes encoding proteins Mmf1p, Hmf1p (or both of them) result in the correlative variation of expression level of the target 12 genes both in the cells with changed phenotype (mmf1 and mmf1 hmf1) and in the cells retaining w.t. shape and growth rate (wt cells, hmf1). Metabolic processes and cellular pathways have been indicated for Mmf1p and Hmf1p based on the different profiles of the expression of 14 genes in mmf1, hmf1 yeast S. cerevisiae cells.